A novel intronic variant in UBE3A identified by genome sequencing in a patient with an atypical presentation of Angelman syndrome

Angelman syndrome (AS) is a genetic neurodevelopmental disorder caused by loss or deficient expression of UBE3A on the maternally inherited allele. In 10–15% of individuals with a clinical diagnosis of AS, a molecular diagnosis cannot be established with conventional testing. We describe a 13‐year‐old male with an atypical presentation of AS, who was found to have a novel, maternally inherited, intronic variant in UBE3A (c.3‐12T>A) using genome sequencing (GS). Targeted sequencing of RNA isolated from blood confirmed the creation of a new acceptor splice site. These GS results ended a six‐year diagnostic odyssey and revealed a 50% recurrence risk for the unaffected parents. This case illustrates a previously unreported splicing variant causing AS. Intronic variants identifiable by GS may account for a proportion of individuals who are suspected of having well‐known genetic disorders despite negative prior genetic testing.     

Looking for the hidden mutation: Bannayan–Riley–Ruvalcaba syndrome caused by constitutional and mosaic 10q23 microdeletions involving PTEN and BMPR1A

The PTEN hamartoma tumor syndrome (PHTS) is caused by heterozygous germline variants in PTEN. Here, we report two unrelated patients with juvenile polyposis, macrocephaly, intellectual disability, and hyperpigmented skin macules. Both patients were clinically suspected for the Bannayan–Riley–Ruvalcaba syndrome (BRRS), a PHTS subentity. By array‐CGH analysis, we identified an interstitial 10q23.1q23.3 deletion in a buccal mucosa sample of Patient 1 that encompassed PTEN, BMPR1A, and KLLN, among others. In contrast, neither sequencing nor array‐CGH analysis identified a pathogenic variant in PTEN or BMPR1A in a blood sample of Patient 2. However, in a surgical specimen of the thyroid gland high‐level mosaicism for a 10q23.2q23.3 deletion was observed. Additionally, the pathogenic PTEN variant c.956_959delCTTT p.(Thr319LysfsTer24) was detected in his thyroid tissue. The frame shift variant was neither detected in the patient's blood nor in his buccal mucosa sample. Low‐level mosaicism for the microdeletion was identified in a buccal swap sample, and reanalysis of the blood sample suggested marginal‐level mosaicism for deletion. The 10q23.2q23.3 deletion mosaicism was also identified in a subsequently resected colonic polyp. Thus, in both cases, the diagnosis of a 10q23 deletion syndrome, which clinically presented as BRRS, was established. Overall, the study expands the BRRS spectrum and highlights the relevance of considering mosaicism in PHTS. We conclude that in all patients with a clear clinical suspicion of PHTS, in which genetic analyses of DNA from blood and buccal swap samples fail to identify causative genetic variants, genetic analyses of additional tissues are recommended.     

Deletions Overlapping VCAN Exon 8 Are New Molecular Defects for Wagner Disease

Wagner disease is a rare nonsyndromic autosomal‐dominant vitreoretinopathy, associated with splice mutations specifically targeting VCAN exon 8. We report the extensive genetic analysis of two Wagner probands, previously found negative for disease‐associated splice mutations. Next‐generation sequencing (NGS), quantitative real‐time PCR, and long‐range PCR identified two deletions (3.4 and 10.5 kb) removing at least one exon–intron boundary of exon 8, and both correlating with an imbalance of VCAN mRNA isoforms. We showed that the 10.5‐kb deletion occurred de novo, causing somatic mosaicism in the proband's mother who had an unusually mild asymmetrical phenotype. Therefore, exon 8 deletions are novel VCAN genetic defects responsible for Wagner disease, and VCAN mosaic mutations may be involved in the pathogenesis of Wagner disease with attenuated phenotype. NGS is then an effective screening tool for genetic diagnosis of Wagner disease, improving the chance of identifying all disease‐causative variants as well as mosaic mutations in VCAN.     

Mosaic KCNJ2 mutation in Andersen–Tawil syndrome: targeted deep sequencing is useful for the detection of mosaicism

Andersen–Tawil syndrome (ATS) is an inherited disease characterized by ventricular arrhythmias, periodic paralysis, and dysmorphic features. It results from a heterozygous mutation of KCNJ2, but little is known about mosaicism in ATS. We performed genetic analysis of KCNJ2 in 32 ATS probands and their family members and identified KCNJ2 mutations in 25 probands, 20 families who underwent extensive genetic testing. These tests revealed that seven probands carried de novo mutations while 13 carried inherited mutations from their parents. We then specifically assessed a single proband and the respective family. The proband was a 9 year old girl who fulfilled the ATS triad and carried an insertion mutation (p.75_76insThr). We determined that the proband's mother carried a somatic mosaicism and that the proband's younger brother also carried the ATS phenotype with the same insertion mutation. The mother, who exhibited mosaicism, was asymptomatic, although she exhibited Q(T)U prolongation. Mutant allele frequency was 11% as per TA cloning and 17.3% as per targeted deep sequencing. Our observations suggest that targeted deep sequencing is useful for the detection of mosaicism and that the detection of mosaic mutations in parents of apparently sporadic ATS patients can help in the process of genetic counseling.     

High frequency of mosaic pathogenic variants in genes causing epilepsy-related neurodevelopmental disorders

PurposeMosaicism probably represents an underreported cause of genetic disorders due to detection challenges during routine molecular diagnostics. The purpose of this study was to evaluate the frequency of mosaicism detected by next-generation sequencing in genes associated with epilepsy-related neurodevelopmental disorders.MethodsWe conducted a retrospective analysis of 893 probands with epilepsy who had a multigene epilepsy panel or whole-exome sequencing performed in a clinical diagnostic laboratory and were positive for a pathogenic or likely pathogenic variant in one of nine genes (CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A, or SCN2A). Parental results were available for 395 of these probands.ResultsMosaicism was most common in the CDKL5, PCDH19, SCN2A, and SCN1A genes. Mosaicism was observed in GABRA1, GABRG2, and GRIN2B, which previously have not been reported to have mosaicism, and also in KCNQ2 and MECP2. Parental mosaicism was observed for pathogenic variants in multiple genes including KCNQ2, MECP2, SCN1A, and SCN2A.ConclusionMosaic pathogenic variants were identified frequently in nine genes associated with various neurological conditions. Given the potential clinical ramifications, our findings suggest that next-generation sequencing diagnostic methods may be utilized when testing these genes in a diagnostic laboratory.     

Somatic mosaic deletions involving SCN1A cause Dravet syndrome

Somatic mosaicism in single nucleotide variants of SCN1A is known to occur in a subset of parents of children with Dravet syndrome (DS). Here, we report recurrent somatic mosaic microdeletions involving SCN1A in children diagnosed with DS. Through the evaluation of 237 affected individuals with DS who did not show SCN1A or PCHD19 mutations in prior sequencing analyzes, we identified two children with mosaic microdeletions covering the entire SCN1A region. The allele frequency of the mosaic deletions estimated by multiplex ligation‐dependent probe amplification and array comparative genomic hybridization was 25–40%, which was comparable to the mosaic ratio in lymphocytes and buccal mucosa cells observed by fluorescence in situ hybridization analysis. The minimal prevalence of SCN1A mosaic deletion is estimated to be 0.9% (95% confidence level: 0.11–3.11%) of DS with negative for SCN1A and PCDH19 mutations. This study reinforces the importance of somatic mosaicism caused by copy number variations in disease‐causing genes, and provides an alternative spectrum of SCN1A mutations causative of DS. Somatic deletions in SCN1A should be considered in cases with DS when standard screenings for SCN1A mutations are apparently negative for mutations.

     

Molecular characterization of a mosaic NIPBL deletion in a Cornelia de Lange patient with severe phenotype

Cornelia de Lange syndrome (CdLS, OMIM #122470, #300590, #610759, #614701, #300882) is a rare neurodevelopmental syndrome characterized by growth retardation, intellectual disability, dysmorphic facial features, multisystem malformations, and limb reduction defects. Wide variability of phenotypes is common among CdLS patients. Mutations in genes encoding either regulators (NIPBL, HDAC8) or subunits (SMC1A, SMC3, RAD21) of the cohesin complex, are altogether found in approximately 65% of CdLS patients.We describe a CdLS patient with classic severe phenotype who was found negative to mutations in the NIPBL and SMC1A genes by DHPLC and direct sequencing. MLPA analysis performed to disclose potential intragenic NIPBL deletions/duplications, suggested a partial deletion which was confirmed by FISH with a BAC clone encompassing the NIPBL region that highlighted asymmetric signals in a fraction of cells (72%). The occurrence of a genomic deletion in mosaic condition was validated by array-CGH analysis. Long-range PCR and sequencing of the junction fragment mapped the telomeric and the centromeric breakpoint within NIPBL IVS1 and IVS32, respectively. Both deletion breakpoints were embedded in a microsatellite region that might be the motif directly mediating this large deletion by an intrachromatid recombination mechanism.Consistent with the molecular analyses, the patient displayed a severe phenotype that was characterized by drastic CdLS clinical signs including premature death. This case provides a second example of mosaicism in CdLS. Despite mitigated by mosaicism, the large intragenic deletion identified in the present case was poorly tolerated due to the high mosaicism level. Based on these data, overlooked cases of mosaicism may lead to underestimated mutation rates of known genes and may also contribute to the clinical heterogeneity of CdLS.     

VHL mosaicism can be detected by clinical next-generation sequencing and is not restricted to patients with a mild phenotype

The identification of Von Hippel-Lindau (VHL) mosaic mutations by conventional Sanger sequencing requires a labour-intensive enrichment step, thus explaining that mosaicism occurrence is underestimated in patients. Nowadays, it is possible to detect mutation in cell sub-populations by next-generation sequencing (NGS). Here, we described a diagnosis strategy using NGS with high coverage in a series of eight patients who were negative for a VHL abnormality by Sanger sequencing and deletion search. In two patients, a mosaic mutation in VHL was detected by NGS. One patient with a 5.7% mutated allele frequency had a severe phenotype and an early disease onset. In conclusion, clinical NGS in an hospital molecular oncogenetics laboratory is an efficient tool to identify VHL mosaic mutation. Its use may improve patient monitoring and genetic counseling.     

A novel mutation of MSX1 inherited from maternal mosaicism causes a severely affected child with nonsyndromic oligodontia

Mutations of MSX1 have been associated with nonsyndromic hypodontia. To seek the causal gene mutation sites in a family with nonsyndromic oligodontia, whole-exome sequencing (WES) was performed to seek the causative locus of the family. The candidate mutation was further identified by Sanger sequencing afterward. Two mutations of MSX1 were found both in the proband and her mother. One novel heterozygous missense mutation (c.C667G, p.R223G) of MSX1 inherited from the asymptomatic mother with mosaic mutation was located in the highly conserved fragment of exon 2. The other was a synonymous mutation (c.C348T, p.G116G) in exon 1, which had been reported. The novel maternal heterozygous missense mutation (c.C667G, p.R223G) was likely to be the major reason for nonsyndromic oligodontia in the family. This is the first mosaic variant that has been recorded of the MSX1 gene. Our study expands the phenotype–genotype correlation associated with MSX1 variants. Our study also suggests that the determination of the mosaicism is essential for precise genetic counseling if a disease appears to arise de novo.     

Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of “de novo” SCN1A Mutations in Children with Dravet Syndrome

The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling.     

Low-level mosaicism in tuberous sclerosis complex: prevalence,clinical features,and risk of disease transmission

PurposeTo examine the prevalence and spectrum of mosaic variant allele frequency (MVAF) in tuberous sclerosis complex (TSC) patients with low-level mosaicism and correlate genetic findings with clinical features and transmission risk.MethodsMassively parallel sequencing was performed on 39 mosaic TSC patients with 170 different tissue samples.ResultsTSC mosaic patients (MVAF: 0–10%, median 1.7% in blood DNA) had a milder and distinct clinical phenotype in comparison with other TSC series, with similar facial angiofibromas (92%) and kidney angiomyolipomas (83%), and fewer seizures, cortical tubers, and multiple other manifestations (p < 0.0001 for six features). MVAF of TSC1/TSC2 pathogenic variants was highly variable in different tissue samples. Remarkably, skin lesions were the most reliable tissue for variant identification, and 6 of 39 (15%) patients showed no evidence of the variant in blood. Semen analysis showed absence of the variant in 3 of 5 mosaic men. The expected distribution of MVAF in comparison with that observed here suggests that there is a considerable number of individuals with low-level mosaicism for a TSC2 pathogenic variant who are not recognized clinically.ConclusionOur findings provide information on variability in MVAF and risk of transmission that has broad implications for other mosaic genetic disorders.     

Grandparental genotyping enhances exome variant interpretation

Trio exome sequencing is a powerful tool in the molecular investigation of monogenic disorders and provides an incremental diagnostic yield over proband‐only sequencing, mainly due to the rapid identification of de novo disease‐causing variants. However, heterozygous variants inherited from unaffected parents may be inadvertently dismissed, although multiple explanations are available for such scenarios including mosaicism in the parent, incomplete penetrance, imprinting, or skewed X‐inactivation. We report three probands, in which a pathogenic or likely pathogenic variant was identified upon exome sequencing, yet was inherited from an unaffected parent. Segregation of the variants (in NOTCH1, PHF6, and SOX10) in the grandparent generation revealed that the variant was de novo in each case. Additionally, one proband had skewed X‐inactivation. We discuss the possible genetic mechanism in each case, and urge caution in data interpretation of exome sequencing data. We illustrate the utility of expanding segregation studies to the grandparent generation and demonstrate the impact on exome interpretation strategies, by showing that objective genotype data can overcome subjective parental report of lack of symptoms.     

Genetic and clinical findings in a Chinese cohort of patients with collagen VI‐related myopathies

Collagen VI‐related myopathy, caused by pathogenic variants in the genes encoding collagen VI, represents a clinical continuum from Ullrich congenital muscular dystrophy (UCMD) to Bethlem myopathy (BM). Clinical data of 60 probands and their family members were collected and muscle biopsies of 26 patients were analyzed. COL6A1, COL6A2 and COL6A3 exons were analyzed by direct sequencing or next generation sequencing (NGS). Sixty patients were characterized by delayed motor milestones, muscle weakness, skin and joint changes with 40 UCMD and 20 BM. Muscle with biopsies revealed dystrophic changes and showed completely deficiency of collagen VI or sarcolemma specific collagen VI deficiency. We identified 62 different pathogenic variants in these 60 patients, with 34 were first reported while 28 were previously known; 72 allelic pathogenic variants in COL6A1 (25/72, 34.7%), COL6A2 (33/72, 45.8%) and COL6A3 (14/72, 19.4%). We also found somatic mosaic variant in the parent of 1 proband by personal genome machine amplicon deep sequencing for mosaicism. Here we provide clinical, histological and genetic evidence of collagen VI‐related myopathy in 60 Chinese patients. NGS is a valuable approach for diagnosis and accurate diagnosis provides useful information for genetic counseling of related families.     

Improved diagnostic yield of neuromuscular disorders applying clinical exome sequencing in patients arising from a consanguineous population

Neuromuscular diseases (NMDs) include a broad range of disorders affecting muscles, nerves and neuromuscular junctions. Their overlapping phenotypes and heterogeneous genetic nature have created challenges in diagnosis which calls for the implementation of massive parallel sequencing as a candidate strategy to increase the diagnostic yield. In this study, total of 45 patients, mostly offspring of consanguineous marriages were examined using whole exome sequencing. Data analysis was performed to identify the most probable pathogenic rare variants in known NMD genes which led to identification of causal variants for 33 out of 45 patients (73.3%) in the following known genes: CAPN3, Col6A1, Col6A3, DMD, DYSF, FHL1, GJB1, ISPD, LAMA2, LMNA, PLEC1, RYR1, SGCA, SGCB, SYNE1, TNNT1 and 22 novel pathogenic variants were detected. Today, the advantage of whole exome sequencing in clinical diagnostic strategies of heterogeneous disorders is clear. In this cohort, a diagnostic yield of 73.3% was achieved which is quite high compared to the overall reported diagnostic yield of 25% to 50%. This could be explained by the consanguineous background of these patients and is another strong advantage of offering clinical exome sequencing in diagnostic laboratories, especially in populations with high rate of consanguinity.     

Structural mapping of GABRB3 variants reveals genotype–phenotype correlations

PurposePathogenic variants in GABRB3 have been associated with a spectrum of phenotypes from severe developmental disorders and epileptic encephalopathies to milder epilepsy syndromes and mild intellectual disability (ID). In this study, we analyzed a large cohort of individuals with GABRB3 variants to deepen the phenotypic understanding and investigate genotype–phenotype correlations.MethodsThrough an international collaboration, we analyzed electro-clinical data of unpublished individuals with variants in GABRB3, and we reviewed previously published cases. All missense variants were mapped onto the 3-dimensional structure of the GABRB3 subunit, and clinical phenotypes associated with the different key structural domains were investigated.ResultsWe characterized 71 individuals with GABRB3 variants, including 22 novel subjects, expressing a wide spectrum of phenotypes. Interestingly, phenotypes correlated with structural locations of the variants. Generalized epilepsy, with a median age at onset of 12 months, and mild-to-moderate ID were associated with variants in the extracellular domain. Focal epilepsy with earlier onset (median: age 4 months) and severe ID were associated with variants in both the pore-lining helical transmembrane domain and the extracellular domain.ConclusionThese genotype–phenotype correlations will aid the genetic counseling and treatment of individuals affected by GABRB3-related disorders. Future studies may reveal whether functional differences underlie the phenotypic differences.     

Parental somatogonadal COL2A1 mosaicism contributes to intrafamilial recurrence in a family with type 2 collagenopathy

The COL2A1 gene encodes the alpha‐1 chain of procollagen type 2. Pathogenic variants in the COL2A1 gene are associated with several different types of skeletal dysplasia collectively known as type 2 collagenopathies. Type 2 collagenopathies have an autosomal dominant inheritance. Some germline or somatogonadal mosaicism cases have been reported. We investigated whether somatogonadal mosaicism occurred in a family with two children suspected of type 2 collagenopathies or related diseases. First, we detected a pathogenic variant in the COL2A1 gene in the two affected children by whole exome sequencing (WES). Next, we performed targeted deep sequencing to their parents without the variant by WES. A low level of COL2A1 mosaicism was revealed in the mother's tissues. We concluded that the mother had somatogonadal mosaicism with the COL2A1 mutation arose in the epiblast, and that the intrafamilial recurrence rate of the disease by the somatogonadal mosaicism was higher than by the germline mosaicism. This report suggests that parental low‐level mosaicism should be evaluated in those parents with children carrying de novo germline mutations and the targeted deep sequencing is useful to detect them.     

The contribution of mosaicism to genetic diseases and de novo pathogenic variants

The contribution of mosaicism to diagnosed genetic disease and presumed de novo variants (DNV) is under investigated. We determined the contribution of mosaic genetic disease (MGD) and diagnosed parental mosaicism (PM) in parents of offspring with reported DNV (in the same variant) in the (1) Undiagnosed Diseases Network (UDN) (N = 1946) and (2) in 12,472 individuals electronic health records (EHR) who underwent genetic testing at an academic medical center. In the UDN, we found 4.51% of diagnosed probands had MGD, and 2.86% of parents of those with DNV exhibited PM. In the EHR, we found 6.03% and 2.99% and (of diagnosed probands) had MGD detected on chromosomal microarray and exome/genome sequencing, respectively. We found 2.34% (of those with a presumed pathogenic DNV) had a parent with PM for the variant. We detected mosaicism (regardless of pathogenicity) in 4.49% of genetic tests performed. We found a broad phenotypic spectrum of MGD with previously unknown phenotypic phenomena. MGD is highly heterogeneous and provides a significant contribution to genetic diseases. Further work is required to improve the diagnosis of MGD and investigate how PM contributes to DNV risk.     

Somatic mosaics in hereditary tumor predisposition syndromes

Historically, it is estimated that 5–10% of cancer patients carry a causative genetic variant for a tumor predisposition syndrome. These conditions have high clinical relevance as they are actionable regarding risk-specific surveillance, predictive genetic testing, reproductive options, and – in some cases – risk reducing surgery or targeted therapy. Every individual is born with on average 0.5–1 exonic mosaic variants prevalent in single or multiple tissues. Depending on the tissues affected, mosaic conditions can abrogate the clinical phenotype of a tumor predisposition syndrome and can even go unrecognized, because it can be impossible or difficult to detect them with routine genetic testing in blood/leucocytes. On the other hand, it is estimated that at least 4% of presumed de novo variants are the result of low-level mosaicism (variant allele frequency <10%) in a parent, while around 7% are true mosaic variants with a higher variant allele frequency, which can sometimes be confused for heterozygous variants. Clonal hematopoiesis however can simulate a mosaic tumor predisposition in genetic diagnostics and has to be taken into account, especially for TP53 variants.Depending on the technique, variant allele frequencies of 2–3% can be detected for single nucleotide variants by next generation sequencing, copy number variants with variant allele frequencies of 5–30% can be detected by array-based technologies or MLPA.Mosaic tumor predisposition syndromes are more common than previously thought and may often remain undiagnosed. The clinical suspicion and diagnostic procedure for several cases with mosaic tumor predisposition syndromes are presented.